The NMDA receptor is the major excitatory amino acid receptor that mediates glutamate transmission in the central nervous system. This receptor has been implicated in neuronal modulation, including long term potentiation in the hippocampus (Collingridge and Singer, TIPS, 1990, 11, 290). It is consequently believed to play a key role in memory acquisition and learning.
The integral channel of the NMDA receptor allows ca.sup.2+ to permeate as well as Na.sup.+ and K.sup.+, and presents at least seven pharmacologically distinct sites. These include a glutamate binding site, a glycine binding site, a polyamine site, a dizocilpine binding site, a voltage dependent Mg.sup.2+ site, a Zn.sup.2+ binding site (Wong and Kemp, Ann. Rev. Pharmacol. Toxicol., 1991, 31, 401) and an ifenprodil binding site (Carter et al., J. Pharmacol. Exy. Ther., 1988, 247, 1222).
The development of potent and selective NMDA receptor antagonists which penetrate into the brain has received considerable attention of late as an attractive strategy for treating and/or preventing conditions which are believed to arise from over-stimulation of neurotransmitter release by excitatory amino acids. Such conditions notably include neurodegenerative disorders arising as a consequence of such pathological conditions as stroke, hypoglycaemia, cerebral palsy, transient cerebral ischaemic attack, cerebral ischaemia during cardiac pulmonary surgery or cardiac arrest, perinatal asphyxia, epilepsy, Huntington's chorea, Alzheimer's disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, Olivo-ponto-cerebellar atrophy, anoxia such as from drowning, spinal cord and head injury, and poisoning by exogenous and endogenous NMDA receptor agonists and neurotoxins, including environmental neurotoxins.
NMDA receptor antagonists may also be useful as anticonvulsant and antiemetic agents, as well as being of value in the prevention or reduction of dependence on dependence-inducing agents such as narcotics.
NMDA receptor antagonists have recently been shown to possess analgesic (see, for example, Dickenson and Aydar, Neuroscience Lett., 1991, 121, 263; Murray et al., Pain, 1991, 44, 179; and Woolf and Thompson, Pain, 1991, 44, 293) and anxiolytic (see, for example, U.S. Pat. No. 5,145,866; and Kehne et al., Eur. J. Pharmacol., 1991, 193, 283) effects, and such compounds may accordingly be useful in the management of pain, depression and anxiety.
Compounds possessing functional antagonist properties for the NMDA receptor complex are stated in WO-A-91/19493 to be effective in the treatment of mood disorders, including major depression, bipolar disorder, dysthymia and seasonal affective disorder (cf. also Trullas and Skolnick, Eur. J. Pharmacol., 1990, 185, 1). Such compounds may consequently be of benefit in the treatment and/or prevention of those disorders.
The association of NMDA receptor antagonists with regulation of the dopaminergic system has recently been reported (see, for example, Werling et al., J. Pharmacol. Exp. Ther., 1990, 255, 40; Graham et al., Life Sciences, 1990, 47, PL-41; Hutson et al., Br. J. Pharmacol., 1991, 103, 2037; and Turski et al., Nature (London), 1991, 349, 414). This suggests that such compounds may thus be of assistance in the prevention and/or treatment of disorders of the dopaminergic system such as schizophrenia and Parkinson's disease.
It has also been reported recently (see Lauritzen et al., Journal of Cerebral Blood Flow and Metabolism, 1991, vol. 11, suppl. 2, Abstract XV-4) that NMDA receptor antagonists block cortical spreading depression (CSD), which may thus be of clinical importance since CSD is a possible mechanism of migraine. The class of substituted 2-amino-4-phosphonomethylalk-3-ene carboxylic acids and esters described in EP-A0420806, which are stated to be selective NMDA antagonists, are alleged thereby to be of potential utility in the treatment of inter alia migraine.
Excitatory amino acid receptor antagonists, including inter alia antagonists of NMDA receptors, are alleged in EP-A-0432994 to be of use in suppressing emesis.
Recent reports in the literature have also suggested a link between the neurotoxicity of certain viruses and the deleterious effects of these viruses on an organism caused by the potentiation of neurotransmission via excitatory amino acid receptors. Antagonists of NMDA receptors may therefore be effective in controlling the manifestations of neuroviral diseases such as measles, rabies, tetanus (cf. Bagetta et al., Br. J. Pharmacol., 1990, 101, 776) and AIDS (cf. Lipton et al., Society for Neuroscience Abstracts, 1990, 16, 128.11).
NMDA antagonists have, moreover, been shown to have an effect on the neuroendocrine system (see, for example, van den Pol et al., Science, 1990, 250, 1276; and Urbanski, Endocrinology, 1990, 127, 2223), and such compounds may therefore also be effective in the control of seasonal breeding in mammals.
A cDNA, encoding a subunit of the rat NMDA receptor and designated NMDA R1, has been cloned by expression cloning (Moriyoshi et al., Nature (London), 1991, 354, 31). When expressed in Xenopus oocytes, this cDNA exhibits the electrophysiological and pharmacological properties expected of an authentic NMDA receptor, although the levels of expression are extremely low. More recently, the existence of several discrete isoforms of the rat NMDA R1 receptor subunit, generated by alternative RNA splicing, has been reported (Sugihara et al., BBRC, 1992, 185, 826). Using both low stringency hybridization and polymerase chain reaction methodologies, four additional rodent NMDA receptor subunit cDNAs have been cloned: .epsilon.1 or NMDA R2A; .epsilon.2 or NMDA R2B; .epsilon.3 or NMDA R2C; and .epsilon.4 or NMDA R2D (see Monyer et al., Science, 1992, 256, 1217; Kutsuwada et al., Nature (London), 1992, 358, 36; Ikeda et al., FEBS Lett., 1992, 313, 34; and Ishii et al., J. Biol. Chem., 1993, 268, 2836). Co-expression in Xenopus oocytes or transiently transfected cells of the NMDA R1 subunit, with any one of the R2A, R2B, R2C or R2D subunits referred to above, gives rise to a more robust NMDA receptor than that constituted by the NMDA R1 subunit alone (Monyer et al., Science, 1992, 256, 1217). Moreover, these four resulting putative NMDA receptors (R1/R2A; R1/R2B; R1/R2C; and R1/R2D) are observed to be pharmacologically and electrophysiologically distinguishable. These data support the hypothesis that a family of NMDA receptor subtypes with distinct pharmacological profiles may exist in the brain through combination of different subunits.
Any of a variety of procedures may be used to molecularly clone human NMDA receptor cDNA. These methods include, but are not limited to, direct functional expression of the human NMDA receptor cDNAs following the construction of a human NMDA receptor containing cDNA library in an appropriate expression vector system. Another method is to screen a human NMDA receptor containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the purified NMDA receptor protein or from the DNA sequence of known NMDA receptor cDNAs. The preferred method consists of screening a human NMDA receptor containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a .sup.32 P-labelled cDNA oligonucleotide-primed fragment of rodent NMDA receptor subunit cDNA. The preferred human cDNA library is a commercially available human hippocampal cDNA library.
It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other brain regions, may be useful for isolating DNA encoding the human NMDA receptor. Other types of libraries include, but are not limited to, cDNA libraries derived from other tissues, cells or cell lines other than human hippocampal cells, and genomic DNA libraries.
Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found, for example, in Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, New York, 2nd edition, 1989).
It is also readily apparent to those skilled in the art that DNA encoding the human NMDA receptor may also be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techiques can be found in Maniatis et al., supra.